<p>HeLa cells growing on cover glass were transfected with pCAGGS encoding wild-type NP-NES3 or its mutants (Φ1, Φ2, Φ3, or Φ4) for 48 h. The cells were then permeabilized with 50 µg/ml digitonin for 5 min on ice. The cytoplasmic components, including NP, were removed by washing (only NP in the nucleus remained). The nuclear export activity of NP was allowed to proceed in the presence of fresh total HeLa cell lysate at 30°C for 1 h (left column). Negative controls were incubated in the absence of total cell lysate (right panel). The cells were then stained with an anti-NP MAb followed by anti-mouse Alexa Flour 488 and Hoechst 333342, and observed under a confocal laser-scanning microscope. The white arrow head indicates nuclear export activity, whereas the yellow arrow head indicates a failure of nuclear export activity.</p
